expression and function of -smooth muscle actin during...

229Research Article

IntroductionThe six actin isoforms found in mammalians constitute afamily of closely related proteins expressed in a tissue-specificway. �- and �-cytoplasmic actins are ubiquitous, and fourmuscle actins with very similar primary sequences [�-skeletal(�-SKA), �-cardiac (�-CAA), �-smooth muscle (�-SMA) and�-smooth muscle actin (�-SMA)] are found in the differentmuscle types. Despite their high similarity, we and others havebeen able to develop specific antibodies for some actinisoforms: �-SMA (Skalli et al., 1986), �-cytoplasmic actin(Gimona et al., 1994), �-SKA (Clement et al., 1999) and �-CAA (Clement et al., 2003; Franke et al., 1996).

In normal myocardium, �-CAA, �-SKA and �-SMA, areco-expressed and the amount of their transcripts has beenshown to vary with species, developmental stage, aging andduring pathological situations (Carrier et al., 1992; Schwartzet al., 1992; Schwartz et al., 1986; Winegrad et al., 1990).During in vivo cardiogenesis, �-SMA marks the onset ofcardiomyocyte differentiation, and as development proceeds, itis sequentially replaced by �-SKA and �-CAA isoforms(Ruzicka and Schwartz, 1988; Woodcock-Mitchell et al.,1988). In the mouse embryo, as the cardiac compartment isformed, the early cardiomyocytes express all �-muscle actinisoforms, with �-CAA being predominantly expressed

throughout development (Sassoon et al., 1988). In normal adultmyocardium, the two sarcomeric actins, �-CAA and �-SKA,are co-expressed and represent the preponderant actin isoforms(Vandekerckhove et al., 1986). The significance of thesetransitions in actin gene expression during myogenesis is stillan open question. In addition, when newborn and adultcardiomyocytes are cultured in vitro, they re-express fetalproteins such as �-SMA, �-SKA, �-myosin heavy chain (�-MHC), and atrial natriuretic factor (ANF) (Eppenberger-Eberhardt et al., 1990; Schaub et al., 1997; van Bilsen andChien, 1993). These genes are also re-expressed during cardiachypertrophy in vivo and represent well-accepted markers ofthis phenomenon.

It has been hypothesized that muscle actin isoforms may berequired to achieve different degrees of myocardialcontractility and several approaches have been used to examinethe developmental and functional significance of these actins:for instance, the targeted expression of �-SMA, the only actinisoform normally absent in the myocardium, in the heart oftransgenic mice results in a hypodynamic heart (Kumar et al.,1997). Knockout mice for �-CAA usually die in the neonatalperiod. However, when �-SMA is expressed under the controlof the cardiac �-MHC promoter, these knockout mice surviveto adulthood, but their hearts remain highly hypodynamic.

Three �-muscle actin isoforms are sequentially expressedduring in vivo cardiac development. �-Smooth muscle actinis first and transiently expressed, followed by �-skeletal andfinally �-cardiac actin. The significance of these transitionsin actin gene expression during myogenesis remains to bedetermined. To understand whether actin isoforms havespecific functions during cardiac development andcardiomyocyte contractility, we have hampered �-smoothmuscle and �-skeletal actin expression and organizationduring embryonic stem cell differentiation towardscardiomyocyte. We show that the sequence of actin isoformexpression displays similar pattern in the in vitro modeland in mouse heart embryogenesis. Treatment with aninterfering fusion peptide containing the N-terminalsequence of �-smooth muscle actin during a time window

preceding spontaneous beating, prevents proper cardiacsarcomyogenesis, whereas �-skeletal actin-fusion peptidehas no effect. Knockdown of �-smooth muscle actin inembryonic stem cells using RNA interference also affectscardiac differentiation. The application of both fusionpeptides on beating embryoid bodies impairs frequency.These results suggest specific functional activities for actinisoforms in cardiogenesis and cardiomyocyte contractility.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/2/229/DC1

Key words: Cardiomyocyte contraction, Antennapedia-fusionpeptide, shRNA, Cytoskeleton, Cardiogenesis, Heart,Sarcomyogenesis

Summary

Expression and function of �-smooth muscle actinduring embryonic-stem-cell-derived cardiomyocytedifferentiationSophie Clément1,*,‡, Michael Stouffs1,‡, Esther Bettiol1,‡, Sandy Kampf1,‡, Karl-Heinz Krause1,‡,Christine Chaponnier2 and Marisa Jaconi1,‡

1Department of Geriatrics, Laboratory of Ageing, Geneva Hospital, Chêne-Bourg, Geneva, Switzerland2Department of Pathology and Immunology, Faculty of Medicine, CMU, Geneva, Switzerland*Author for correspondence (e-mail: [email protected])‡Present address: Department of Pathology and Immunology, CMU, 1, Rue Michel-Servet, 1211 Geneva 4, Switzerland

Accepted 14 November 2006Journal of Cell Science 120, 229-238 Published by The Company of Biologists 2007doi:10.1242/jcs.03340

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More recently, Martin et al. have shown that the substitutionof �-SMA for �-CAA in isolated cardiac fibers alters the actininteraction with its partners in the myofilament (troponin andmyosin) (Martin et al., 2002). In addition, myofilamentscontaining �-SMA display a decreased sensitivity to Ca2+

(Martin et al., 2002). Thus, even if �-SMA can substitute �-CAA, it cannot completely rescue the heart function. However,perfused hearts isolated from BALB/c mice, which naturallyexpress high levels of �-SKA (Garner et al., 1986), showincreased levels of contractility compared with other strains ofmice (Hewett et al., 1994). Altogether, these observationssupport the assumption that very few amino acid differencesbetween muscle actin isoforms can have major functionalconsequences. In rat cardiomyocytes, functional heterogeneitybetween the different actin isoforms has been investigated bymonitoring the consequences of their ectopic expression (vonArx et al., 1995). Incorporation of �-SMA was observed instress fiber-like structures and sarcomeres, contrary to the threeother muscle actins, which exclusively displayed a sarcomericincorporation. Expression of cytoplasmic actins induceddramatic phenotypic changes and cessation of beating of adultrat cardiomyocytes (ARC). Albeit, the beating activity of ARCwas not hampered by the �-SMA, �-SMA effect was notreported. The N-terminus, where the main differences arelocated, has been directly implicated in the binding of severalactin binding proteins such as gelsolin (Sutoh and Yin, 1989)or troponin I (Lehman et al., 2001). The physiologicalsignificance of such domain has been recently validated in thecase of �-SMA in myofibroblasts. Intracellular delivery of the�-SMA fusion peptide (FP) SMA-FP, containing the N-terminal sequence AcEEED of �-SMA fused to the 16-amino-acid third domain of the Antennapedia homeodomain (pAntp)(Derossi et al., 1994) abolished �-SMA staining in stress fibers(Hinz et al., 2002), leading to a significant decrease ofmyofibroblast contractility both in vitro and in vivo (Hinz etal., 2002). We therefore decided to expand these investigationsto other actin isoforms, in particular to understand theirfunction during cardiac differentiation.

Mouse embryonic stem (ES) cells provide a uniqueexperimental model to study the regulation of cardiomyocytegrowth and differentiation in vitro. ES cells are derived fromthe inner cell mass of the blastocyst and can be maintained inculture as a self-renewing pluripotent population in thepresence of leukemia inhibitory factor (LIF) (Robertson, 1987;Smith et al., 1988). ES cells differentiate in vitro in a broadrange of specific cell types of all three germ layers includingcardiomyocytes. Cultured within embryoid bodies (EBs), EScells recapitulate the development of cardiomyocytes fromearly cardiac precursors to terminally differentiated cells. Theappearance of spontaneously beating cardiomyocytes isobserved after 1 week of culture.

Using the ES cell differentiation model and combiningdifferent analytical and technical approaches (e.g. specificantibodies, fusion peptides), we provide here the first clues inthe understanding of the specific functions of �-SMA and �-SKA, the two ‘non heart-typical’ actin isoforms expressedduring heart development.

ResultsActin isoform expression during mouse developmentSince most of the information available in the literature on �-

actin expression during cardiogenesis resulted from mRNAanalyses, we have carried out careful examination of the threemuscle actin isoforms expression at the protein level. Weperformed immunochemistry using specific anti-actinantibodies during embryogenesis and post natal development.Fig. 1 shows that, at day 9.5 post coitum (E9.5), all �-actinisoforms are present in the heart, �-SKA being expressed onlyin a few cells (Fig. 1Ab, arrows). At E13, the expression levelsof �-SMA, �-SKA and �-CAA were comparable (Fig. 1Ad-f); the area of positive staining represented approximately 65%(Fig. 1B). At E17, �-SMA started to be downregulated (Fig.1Ag; Fig. 1B) and 2 weeks after birth, it was only expressedin smooth muscle cells within the vessels (Fig. 1j; Fig. 1B). Atthis time, �-SKA was present in a subpopulation ofcardiomyocytes (10.3±2.5%), as previously shown in otherspecies (Clement et al., 1999; Clement et al., 2001; Suurmeijeret al., 2003).

Actin isoform expression during ES cell differentiationIt has been extensively shown that ES cell differentiationmimiks in vivo cardiac development (Sachinidis et al., 2003).To ensure that this was true for actin isoform expression andto validate ES cell differentiation as an appropriate model tostudy their function, we established the temporal expression ofactin isoforms by triple staining immunofluorescence onembryoid bodies (EB) at day 6, 8, 12 and 15. Western blot aswell as triple staining immunofluorescence (Fig. 2) illustratethat the sequence of isoactin expression closely correlated withthe one previoulsy observed at mRNA level (Ng et al., 1997).Indeed, �-SMA is the first actin isoform, appearing at day 6 inthe cells of the outer layer of EBs (Fig. 2Aa; Fig. 2C). At day8, �-SKA and �-CAA started to be expressed within thebeating areas (Fig. 2Ae-h; highlighted by dashed white lineson overlay pictures). At high magnification, we observed thatall three isoforms were organized in striations but that theirexpression appeared heterogeneous within the beating area(Fig. 2B, inset). �-SMA is also highly expressed in thefibroblast-like cells that border the EB, as previously described(Ng et al., 1997). From day 12, �-SMA is markedlydownregulated in the cardiomyocytes (Fig. 2Ai,m) but not inthe fibroblast-like cells. This downregulation, however, wasonly visible by immunofluorescence, because western blottinganalyses were performed with protein extracted from all typesof cells present in EBs and it was impossible to discriminatebetween cell types (e.g. smooth muscle cells or myofibroblastsfor �-SMA, or skeletal muscle cells for �-SKA).

Thus, the expression of actin isoforms in ES-cell-derivedcardiomyocytes closely follows the timing of expressionobserved by immunohistochemistry during cardiacdevelopment in vivo. These results validate the ES celldifferentiation system as a suitable in vitro model to study thefunction of actin isoforms in cardiogenesis.

Analysis of actin isoform function during ES celldifferentiationTo understand how the expression of �-SMA and �-SKA isimportant to achieve correct terminal differentiation of ES-cell-derived cardiomyocytes, we have inhibited their function withfusion-peptides (FPs); SMA-FP and SKA-FP contain Ac-EEED and Ac-DEDE, respectively, at the N-terminus of thecell-penetrating vector pAntp-Pro50. As FPs precipitated in

Journal of Cell Science 120 (2)

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presence of fetal calf serum (FCS), whichis usually present in ES celldifferentiation media, we haveestablished culture conditions in thepresence of knockout serum (KO serum,Invitrogen; serum replacement withdefined formulation initially designed tosupport the growth of undifferentiated EScells). Using such culture conditions, wecould confirm that cardiac differentiation– identified by the appearance ofspontaneous beating – proceedednormally (59.9±4.2% beating EBs at day8 when cultured in presence of FCScompared with 58.3±4.1% beating EBscultured in presence of KO serum). Toensure that FP efficiency was preservedin KO serum, we have successfully testedthese culture conditions onmyofibroblasts (cells expressing a highlevel of �-SMA; data not shown).

We first investigated the effects of 10and 50 �g/ml SMA-FP or SKA-FP ondifferentiating EBs that were treatedtwice a day during a window of time,preceding the appearance ofspontaneously beating cardiomyocytes –namely from day 6 to day 8 (Fig. 3A). Atday 8, SMA-FP significantly decreasedthe percentage of beating EBs in a dose-dependent manner (Fig. 3B). Viability ofcells within EBs estimated by TrypanBlue dye exclusion technique assay wasnot affected by FP treatment (10.5±2.1% dead cells inuntreated EBs vs 9.9±3.4% dead cells in SMA-FP-treatedEBs). Interestingly, SMA-FP specifically impaired theformation of myofibrils as observed by the non-organized �-actinin pattern (Fig. 3Da compared with b, inset). Although �-SKA was expressed in EBs during this developmental stage,SKA-FP did affect neither beating (Fig. 3B) nor sarcogenesis(Fig. 3Ea compared with b, inset). As expected, the proteinexpression of the different actin isoforms was not affected bythe treatment with FPs (Fig. 3C). This observation is inaccordance with our previous reports (Clement et al., 2005;Hinz et al., 2002) showing that FPs interfere with actin isoformorganization but not with their expression; the disappearanceof immunostaining being explained by the fact that solubleactin (not organized into structures such as myofibrils) is not

stained by the antibody because it is diffusely distributedthroughout the cytoplasm.

To confirm the role of �-SMA during cardiac differentiation,we have designed small interference RNAs (siRNAs; seeMaterials and Methods and Fig. 4A) specific to this isoactin.We first tested the efficacy of these siRNAs on mouse lungfibroblasts, known to differentiate in culture intomyofibroblast-like cells containing high levels of �-SMA(Dugina et al., 1998; Xu et al., 1997). As shown in Fig. 4B, allthree siRNAs (siSMA1-3) reduced �-SMA expression asvisualized by immunofluorescence, whereas control siRNAhad no effect. None of the other actin isoforms (�- and �-cytoplasmic actins) expressed in these cells were affected bysiSMAs (not shown). All results were similarly reproducedwith the two other siSMAs, with siSMA3 showing the most

Fig. 1. Distribution and quantitativeevaluation of actin isoforms during heartdevelopment. (A) 4-�m sections of embryosat E9.5 (a-c), E13 (d-f) and E17 (g-i), andhearts from 2-week-old mice (j-l) were usedfor immunochemistry with anti �-SMA(a,d,g,j), anti �SKA (b,e,h,k) and anti �-CAA (c,f,i,l) antibodies. Bar, 100 �m.(B) The percentage of �-SMA-positive, �-SKA-positive and �-CAA-positivecardiomyocytes was calculated using thesoftware KS400.

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pronounced knockdown effect (Fig. 4Be). Therefore, we onlyshowed experiments performed with siSMA3.

To investigate the capacity of �-SMA-knockdown ES cellsto differentiate in functional cardiomyocytes, stable siSMA-expressor ES cells were generated (referred to as siSMA3-ESC). Previous work by Tang et al. has proven the feasibilityof U6-promoter-driven shRNA expression in ES cells (Tanget al., 2004). Undifferentiated siSMA3-ESC continued tomaintain typical ESC morphology (growth in compactcolonies; data not shown). Oct-4 expression, a marker of stem

cell pluripotency (Fig. 5A), as well as cellproliferation assessed by FACS analysis (Fig.5B) in undifferentiated siSMA3-ESC wascomparable with those of wild-type andsiControl-ES cells. At day 6 of culture, �-SMA content within EBs was markedlyreduced when compared with controls,whereas �-SKA and �-cytoplasmic actinexpression (Fig. 5C, lanes 4-6) was notaffected. Thus, siSMA3 could selectivelyrepress �-SMA expression. During cardiacdifferentiation of ES cells into EBs, �-SMAdownregulation resulted in an impairedactivity of EB beating (Fig. 5D).Approximately 80% of EBs expressingsiSMA3 did not feature any contracting fociat day 8 of culture. Similar results wereobtained with the two other siSMAsdescribed above (data not shown). At day 12,however, such effect on beating becameundetectable (data not shown). The reason forthis loss of siSMAs effect remains to bedetermined. Several hypotheses can beraised. The effect could be due to (1) asilencing of the U6 promoter beyond day 6,(2) a limiting amount of siRNA comparedwith the amount of endogenous �-SMAexpressed in the cells or, (3) the lack of �-SMA in cardiomyocytes induces a delay in

the differentiation process from which cardiomyocytes caneventually recover.

To assess whether the blockade of differentiation was due toa decreased commitment of the cardiac progenitor cells, wemeasured by real-time reverse transcriptase (RT)-PCR theexpression of early transcription factors involved in cardiacdetermination, namely Nkx-2.5 and MEF2C. When comparedwith controls, downregulation of �-SMA led to a 1.6-fold and2.2-fold decrease of Nkx-2.5 and MEF2C, respectively, at day5 (Fig. 5E).


Fig. 2. Temporal and spatial distribution of actinisoforms during ES cell differentiation. (A) EBsfixed at different time points (6, 8, 12 and 15 days)were triple-stained with antibodies against all threeanti-actin isoforms (�-SMA, green; �-SKA, blue;�-CAA, red). In overlay images (d, h, l and p),beating areas are highlighted by a dashed whiteline. Bar, 200 �m. (B) Overlay image of amagnified beating area at day 8 co-stained with �-SMA (green), �-SKA (blue) and �-CAA (red)antibodies. Inset, magnified part of the beatingarea. Bar, 20 �m. Images were acquired with aconfocal microscope using either 10� (A) or 63�oil immersion objectives (B). (C) Whole-cellextracts of undifferentiated ES cell (D0) and EBsat days 6, 7, 8, 12 and 15 were analyzed by SDS-PAGE and immunoblotted with anti-SMA, anti-SKA and anti-CAA antibodies. (D) Schematicrepresentation of expression of actin isoformduring EB differentiation. Width of each barcorrelates with the qualitative changes of the threeactins over time.

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Analysis of actin isoform function on cardiomyocytecontractilityWe then investigated the effect of the peptides on cellcontractility by assessing beating frequency in untreated versustreated EBs. As previously shown for other cell types(Chaponnier et al., 1995; Hinz et al., 2002), SMA-FP and SKA-FP when applied on differentiated EBs for 2 hours at day 8 (Fig.6A and supplementary material Fig. S1A), specifically lead tothe almost complete disappearence of �-SMA and �-SKAimmunodetection, respectively (supplementary material Fig.S1B). We have recently established that this lack ofimmunostaining is due to the blocking of actin incorporation intofilamentous structures by fusion peptides (Clement et al., 2005).Regarding cardiomyocyte contractility, we have found that

application of SKA-FP for 2 hours induced a 1.5-fold decreaseof the beating frequency (Fig. 6B), whereas a 2-hour treatmentwith SMA-FP did not change beating frequency (Fig. 6B) but,rather recurrently, affected the regularity of beating, i.e.occurrence of temporary pauses characteric of arrhythmia (Fig.6Cb compared with a). To quantify these observations, werecorded 20-second-long movies and obtained plots directlyrepresentative of the beating activity (Fig. 6C, see Materials andMethods). Using this method, we could visualize the decreaseof frequency (Fig. 6Cd compared with c) and chaotic rhythms(Fig. 6Cb compared with a) induced by SKA-FP and SMA-FP,respectively. Fourier transformation was carried out with theobjective to analyze these spectral data. The integration of thepeaks representative of irregular beating clearly showed thatSMA-FP treatment increased the index of arrhythmia fourfoldcompared with control conditions (Fig. 6D).

DiscussionOur results suggest that �-muscle actin isoforms that aresequentially expressed during cardiac differentiation playdifferent functional roles in this process. The reliability of ourmodel is supported by the observation that the expression of�-muscle actin isoforms exhibits a similar temporal sequenceduring in vitro ES cell differentiation and in vivo heartdevelopment. Owing to our newly developed specificantibodies, we have extended the knowledge concerning theexpression of the three �-isoactins. Previously, investigationshave been carried out mostly at the mRNA level during mouseheart development (Lyons et al., 1991; Sassoon et al., 1988)and ES cell differentiation (Ng et al., 1997).

�-SMA is the first isoform to be expressed in the peripheralcells of the spread EBs and, at day 8, all three isoforms aresimultaneously present in beating areas. Nevertheless, theexpression of these three proteins appeared to be irregularwithin the beating area indicating such that, at this point, allcardiomyocytes did not have the same expression pattern ofactin isoforms. The notion of cardiomyocyte heterogeneity isin agreement with the fact that, in EBs, different cardiac celltypes can be produced (Maltsev et al., 1993).

Here, we have focused on the physiological relevance ofthe tight regulation of �-SMA during cardiac differentiation.

Fig. 3. Differential effect of SMA-FP and SKA-FP on beating andmyofibril organization. (A) Schematic representation of the protocolused to treat EBs with FPs twice a day with 5 or 10 �g/ml of SMA-FP, or SKA-FP from days 6 to 8. At day 8, the capacity of the treatedES cells to differentiate into fully differentiated cardiomyocytes wasassessed by determining the appearence of spontaneous beating.(B) Percentage of beating EBs at day 8 in untreated EBs (white bars),SMA-FP-treated (striped bars) and SKA-FP (black bars) treated EBs.Error bars represent s.e.m. of a total of five independent experiments(**P�0.001). (C) Western blot analysis of untreated EBs (lanes 1)and EBs treated with 10 �g/ml SMA-FP (lanes 2) or 10 �g/ml SKA-FP (lanes 3). Proteins were immunoblotted with anti-total actin(1C4), anti-SMA, anti-CAA and anti-SKA antibodies. (D) 3Dreconstruction of confocal microscopy images of untreated EBs (a)and SMA-FP-treated EBs (b) fixed at day 8 and double-stained withanti-� actinin (red) and anti-SMA (green) antibodies. (E) 3Dreconstruction of confocal microscopy images of untreated (a) andSKA-FP treated (b) EBs fixed at day 8 and double-stained with anti-� actinin (red) and anti-SKA (green) antibodies. Bars, 10 �m. Insetsin b, magnification of the myofibrils.

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Our results, using both SMA-FP and siRNAs underline theimportance of transient �-SMA expression during cardiacdifferentiation. Hindrance of expression and organization ofthis protein blocked the differentiation process, as reflectedby the decrease in the percentage of beating EBs. This effectmay be explained by three non-exclusive mechanisms. (1) �-SMA could constitute a scaffold for contractile proteinorganization during myofibrillogenesis. The fact that SMA-FP specifically impairs the formation of myofibrils is anargument in favor of this notion, as previouly hypothesized(Clement et al., 2001; Ehler et al., 2004). (2) �-SMA couldbe a major contributor to the production of cellular tension.The implication of �-SMA in cell tension production is wellaccepted (Hinz et al., 2001; Hinz and Gabbiani, 2003; Hinzet al., 2002). In addition, it has been described that mechanicalstimuli profoundly affect cardiomyocyte differentiation (Henget al., 2004). Factors responsible for ES cell commitment toa cardiovascular fate are still poorly understood; nevertheless,hemodynamic fluid forces have been shown to play animportant role during cardiomyogenesis, and loss of shearstress results in the formation of an abnormal cardiac chamberand valve formation (Illi et al., 2005). It is conceivable thatthe absence of �-SMA leads to reduced cell tension and,consequently, to a blockade of cardiogenesis. (3) We showedthat a downregulation of cardiac transcription factors knownto be implicated in cardiac differentiation (Nkx2.5 and MEF2-

C) correlates with the expression of siSMAs.Growing evidence in the literature suggest arelationship between the perturbation ofactin cytoskeleton organization and alteredgene expression (Mack et al., 2001; Posernet al., 2002; Sotiropoulos et al., 1999). In ourmodel of cardiac differentiation, such a rolein the control of gene transcription may beattributed to �-SMA expression andpolymerization.

Our results also show that activities of �-SKA and �-SMA differentially influencecardiomyocyte rythmicity. Once thecardiomyocytes spontaneously contract, SKA-FP decreases cardiomyocyte beating frequencywhereas SMA-FP induces arrhythmia. Theeffect of SKA-FP is in accordance with reportssuggesting that �-SKA increases the contractileproperty of these cells (Clement et al., 2005;Hewett et al., 1994; Suurmeijer et al., 2003).

More complex is the interpretation of the SMA-FP effect. Giventhat ES cells can form an organized, functional cardiacconduction system in vitro (White and Claycomb, 2005), atempting explanation for this arrhythmia induction would be thatpace-maker cells are preferentially affected by SMA-FP. Anobservation favoring this idea is that, during rat heartdevelopment, expression of �-SMA persists longer in theventricular conduction system, making it a convenient marker forthe ventricular conduction system in the fetal heart (Ya et al.,1997). Nevertheless, given that not all cardiac cells within beatingareas in EBs express �-SMA, it is possible that hindrance by cellcontractility in only the �-SMA-positive fraction of cellscontributes to the disorganization of the electrical conduction ofthe signal.

An interesting area for future investigations would be toenlarge this approach to an in vivo model. �-SMA-null micehave been produced (Schildmeyer et al., 2000) and even thoughthey apparently did not suffer from cardiac problems leadingto premature death (they appeared to have no difficulty feedingor reproducing), further and specific investigations on heartfunctionality (in particular possible arrhythmia) would be ofgreat interest.

In conclusion, our results shed some light on �-SKA and �-SMA functions during cardiomyocyte differentiation and oncell rythmicity. In addition, this work confirms that the isoactinN-terminus is functionally crucial, as recently suggested for �-


Fig. 4. Design of shRNAs and their effect on �-SMA expression in lung fibroblasts (A) Schematicrepresentation of shRNA constructs targeting �-SMA gene (shSMA1-2-3). A shRNA sequenceinactive against all actin isoforms was used ascontrol (shCont). The resulting entry vectors werethen recombined into pLenti6/BLOCK-iTTM

RNAi Vector. The loop sequence CGAA isindicated in red. (B) Untransduced mouse lungfibroblasts (a), and mouse lung fibroblaststransduced with shCont (b), shSMA1 (c), shSMA2(d) and sh-SMA3 (e) were stained for �-SMA(green). DAPI (blue) was used to stain nuclei. Bar,20 �m.

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SMA (Hinz et al., 2002) in myofibroblats. The results shownhere further demonstrate that the N-terminal sequences of �-SMA and �-SKA isoforms have a major and specific effect ontheir function.

Materials and MethodsES cells culture and differentiationMouse ES cells CGR8 (European Collection of Cell Cultures Salisbury, Wiltshire,UK) were cultured in BHK21 medium (Gibco, Invitrogen, Basel, Switzerland)supplemented with non-essential amino acids, pyruvate, �-mercaptoethanol,glutamine, penicillin-streptomycin, 10% fetal calf serum (FCS, Gibco) and LIF-conditioned medium in a humidified 5% CO2 atmosphere at 37°C, and maintainedat less than 70% confluency to keep an undifferentiated phenotype (Li et al., 2002;Meyer et al., 2000). The differentiation of ESC was performed by the hangingdrop method (Maltsev et al., 1994). In brief, EBs were formed for 2 days in

hanging drops (450 cells/20 �l) in differentiation medium (BHK21, as describedabove), containing 20% FCS (Hyclone, Logan, UT) and lacking LIF. After 4 daysin suspension, cultured EBs were plated on gelatin-coated 24-well plates orcoverslips (Meyer et al., 2000). The number of EBs that contained beatingcardiomyocytes was counted under a phase-contrast microscope at day 8 ofdifferentiation.

Treatment with fusion-peptides (FPs)The fusion peptides SMA-FP and SKA-FP – containing Ac-EEED and Ac-DEDE,respectively, at the N-terminus of the cell-penetrating vector pAntp-Pro50 (Derossiet al., 1994) – were synthesized to a purity of 95% (UCB Bioproducts, Belgium).The FPs were administrated either to differentiating EBs before the onset of beatingor to contracting EBs. In the first case, FPs were added twice a day (9 am and 6pm) at concentrations of 10 or 50 �g/ml at day 6 and 7 of differentiation. Percentageof beating EBs was estimated at Day 8 (9 am). In the second set of experiments,beating EBs were treated for 2 hours with 50 �g/ml FPs at day 8.

Fig. 5. Effect of shRNAtargeting �-SMA in ES cells.(A) Proteins of undifferentiatedcells (lanes 1 and 4,untransduced ES cells; lanes 2and 5, ES cells transduced withlentivectors expressing shCont;lanes 3 and 6, ES cellstransduced with lentivectorsexpressing shSMA3) weresubmitted to SDS-PAGE(Coomassie Blue, lanes 1-3),transferred onto nitrocelluloseand blotted with anti-Oct-4, amarker of pluripotenciality(lanes 4-6). (B) Cell cycleanalysis by FACS cytometry ofpropidium iodure labeleduntransduced (top), shCont(middle) and shSMA3(bottom) ESC. (C) Proteins ofEBs at day 6 (lanes 1 and 4,untransduced ESC; lanes 2 and5, transduced with lentivectorsexpressing shCont; lanes 3 and6, ES cells transduced withlentivectors expressingshSMA3) were submitted toSDS-PAGE (lanes 1-3;Coomassie Blue staining) andimmunoblotted with anti-�-SMA, anti-�-SKA and anti-�-cytoplasmic actin antibodies(lanes 4-6). (D) The percentageof beating EBs at day 8 wasassessed under all threeconditions. Bars represents.e.m. of five independentexperiments (**P� 0.001).(E) Impact of �-SMAdownregulation on theexpression of cardiactranscription factors Nkx2.5(black bars) and MEF2C(white bars) was evaluated byreal-time RT-PCR. Barsrepresent s.e.m. of threeindependentexperiments.*P�0.05,**P�0.001.

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Electrophoretic and immunoblot analysisFor immunoblotting, cells were thoroughly scraped from culture dishes in samplebuffer (62.5 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol,50 mM DTT, 0.01% Bromophenol Blue). Total cell lysates were run on 10% SDS-minigels (Bio-Rad Laboratories, Glattbrugg, Switzerland) (Laemmli, 1970) andelectroblotted onto nitrocellulose (Towbin et al., 1979). Nitrocellulose membraneswere incubated with anti-SMA [�sm-1 (Skalli et al., 1986)], anti-SKA (Clement etal., 1999), anti-�-CAA (Clement et al., 2003), anti-total actin [Mouse mAb (clone1C4; Chemicon International, Temecula, CA)], anti-Oct4 antibodies (Santa CruzBiotechnology Inc., Santa Cruz, CA) diluted in Tris-buffered saline (TBS)containing 3% BSA and 0.1% Triton X-100 for 2 hours at room temperature. Afterthree washes with TBS, a second incubation was performed with horseradish-

peroxidase-conjugated affinity-purified goat anti-rabbitIgG or anti-mouse IgG (Jackson ImmunoResearchLaboratories, West Grove, PA) at a dilution of 1:10,000 inTBS, containing 0.1% BSA and 0.1% Triton X-100.Peroxidase activity was developed using the ECL westernblotting system (Amersham, Rahn AG, Zürich,Switzerland), according to the manufacturer’s instructionsand blots were scanned (Arcus II; Agfa, Mortsel,Belgium).

Indirect immunofluorescence,immunohistochemistry and confocal laserscanning microscopyFor immunofluorescence staining, cells were fixed in 3%paraformaldehyde in PBS for 10 minutes at roomtemperature followed by three washes with PBS and thenpermeabilized with 0.3% Triton X-100 in PBS for 10minutes. Subsequently, cells were stained with thefollowing primary antibodies: anti-�sm-1 (IgG2a), anti-SKA (rabbit polyclonal), anti-CAA [rabbit polyclonal(Clement et al., 2003) or mouse mAb (IgG1), developedin C.C.’s laboratory (unpublished data)] and mouse anti-�-actinin (IgG1, Sigma), diluted in PBS-Tween 0.1% for

1 hour at room temperature. Samples were then incubated with FITC-conjugatedanti-mouse antibodies (IgG2a specific; Southern Biotechnology, Birmingham, AL);Rhodamine-conjugated anti-mouse antibodies (IgG1 specific; SouthernBiotechnology); CY5-conjugated anti-rabbit Ig (Jackson ImmunoResearchLaboratories) and a 1:1000 dilution of the nuclear dye TOTO-III (Molecular Probes,Inc., Eugene, OR), for 1 hour at room temperature. After washing in PBS, cellswere mounted in polyvinyl alcohol (PVA) as described by Lennette (Lennette,1978). Images were acquired using a confocal microscope (LSM510, Carl Zeiss,Oberkochen, Germany, using either a 10� or a 63� oil immersion objective).

For immunochemistry, embryos from BALBc mice (at stage E9.5, 13, 17) and 2-week-old mice hearts were fixed in 10% neutral buffered formol and embedded inparaffin. For immunochemistry, 4-�m sections were used with anti-�-sm-1, anti-�-SKA and anti-�-CAA antibodies. Immunoperoxidase staining was performedessentially as described previously (Clement et al., 1999). After staining, imageswere acquired using an Axiophot microscope (Carl Zeiss) equipped with a highsensibility color camera (Axiocam, Carl Zeiss). A set of images was analyzed toassess the percentage of the isoactin-positive cardiomyocyte area using the KS400software (Kontron System, Zeiss Vision, Oberkochen, Germany) as previouslyreported (Clement et al., 1999).

Recording and analysis of cell contractilityRecording of 20-second-long phase-contrast movies of beating EBs at day 8 waswith a Nipkow microscope equipped with Ultraview software (PerkinElmer, Boston,MA). The effect of FP treatment was evaluated on EBs with a frequency ofapproximately 100 beats/minute. Regions of interest were drawn at the peripheryof beating clusters. Beating of these regions generated movements detectable byvariations in the gray level, which were recorded over time with Ultraview software.The resulting frequency plots accurately represent the beating activity. Beating-ratespectra were obtained by fast Fourier transformation of the frequency plots. Lowestand highest frequencies were excluded in favor of the middle range frequencies,


Fig. 6. Effect of FPs on cardiomyocyte beatingactivity. (A) SMA-FP and SKA-FP (50 �g/ml)were applied on differentiated EBs at day 8 for2 hours. (B) The frequency of beating wasestimated by counting under the microscope thenumber of beats per 30 seconds of about 50EBs during a 2-hours period without anytreatment (�) or following a treatment withSMA-FP (�) or SKA-FP (�). (C) 20-secondmovies of beating EBs were recorded and thevariation of grey level in regions at theperiphery of EB was calculated over time. Plotsare representative of the EBs beating activitybefore (a and c) and after 2 hours treatmentwith 50 �g/ml of either SMA-FP (b) or SKA-FP (d). (D) To obtain beating rate spectrums ofthe frequency plots, Fourier transformation wasperformed on plots shown in C (see Materialsand Methods for details). Numbers on the y-axis represent an ‘index of arrhythmia’.

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which are the most representative for beating rates. The main peak of the spectrum,corresponding to the basal frequency, was removed so that only the informationabout arrhythmic beating remained. Spectral analyses and beating-rate variabilitywere compared between untreated and FP-treated EBs. A similar power spectralanalysis of rate fluctuations has been established by Akselrod et al. (Akselrod et al.,1981).

Short hairpin RNA and Gateway lentiviral systemWe designed siRNA for �-SMA using the siRNA selection program developed atthe Whitehead Institute (custom AAN19). Best candidates were selected accordingto the criteria associated with siRNA functionality identified by Reynolds et al.(Reynolds et al., 2004) and submitted to a BLAST search against the mouse genometo ensure the selective targeting of the SMA gene. The chosen siRNAs, 21-base-pair (bp) homologs to either the coding region (siSMA3, bp 1135-1157) or the 3�UTR sequence (siSMA1, bp1310-1332 and siSMA2, bp 1306-1328) of mouse �-SMA (accession number BC064800) were converted to short hairpin RNAs(shRNAs) using the BLOCK-iTTM RNAi designer (Invitrogen). They were thencloned in the pENTRTM/U6 vector (Invitrogen) according to the manufacturer’sinstructions. A shRNA sequence inactive against all actin isoforms was used ascontrol. The resulting entry vectors were then recombined with pLenti6/BLOCK-iTTM RNAi vectors (Invitrogen) using the Gateway® LR plus clonase enzyme mix(Invitrogen).

Lentivector production and transductionThe lentivector particles were produced by transient transfection in HEK 293T cellsas previously described (Dull et al., 1998). The lentivector-containing supernatantwas collected after 72 hours, filtered through a polyethersulfone membrane (poresize 0.45 �m) and concentrated 120-fold by ultracentrifugation (25,000 g, for 90minutes at 4°C). The pellet was resuspended in complete cell culture medium andsubsequently added to the target cells. Estimated titers of the concentratedlentivector were between 5�107 and 1�108 transducing units per ml. ES cells (104

cells/well in six-well plates) or mouse lung fibroblasts (104 cells/well; kindlyprovided by C. Barazzone) (Pagano et al., 2005) were seeded on gelatin-coated six-well plates and transduced the next day. Two days later, cells were split into gelatin-coated culture dishes. Three days after transduction, 7.5 �g/ml blasticidin was addedto the culture medium of ES cells and the selection was maintained for 6 days.

RNA isolation, reverse transcription and real-time quantitativePCRTotal RNA was isolated from EBs at day 5 using TRIzol reagent (Invitrogen).Reverse transcription (RT) was performed in a 20-�l-mixture containing 1 �g oftotal RNA, 50 �M of random hexamers and 200 units of Superscript II (Invitrogen).The reaction was incubated at 42°C for 90 minutes and the volume was then adjustedto 30 �l. The nucleotide sequences of the PCR primers were: MEF2C forward, 5�-CCTACATAACATGCCGCCATCT-3�; MEF2C reverse, 5�-GTGGTACGGTCTCC-CAACTGA-3�; Nkx2.5 forward, 5�-GGATAAAAAAGAGCTGTGCGC-3�; Nkx2.5reverse 5�-GGCTTTGTCCAGCTCCACTG-3�; �-tubulin forward, 5�-AGA-CAACTTCGTTTTCGGTCAGT-3�; �-tubulin reverse, 5�-CCTTTAGCCCAGTT -GTTGCCT-3�.

To avoid amplification of genomic DNA, primers were designed to be intron-spanning. Real-time quantitative PCR was performed using a TaqMan rapid thermalcycler (ABI Prism 7700) in 25 �l reaction mixtures containing 12.5 �l of SYBRGreen PCR Master mix (Applied Biosystems, Foster City, CA), appropriate primerconcentration and 1 �l of cDNA. The relative cDNA concentrations wereestablished from a standard curve using sequential dilutions of corresponding PCRfragments. The amplification program included the initial denaturation step at 95°Cfor 10 minutes, and 40 cycles of denaturation at 95°C for 10 seconds, annealing andextension at 60°C for 1 minute. Fluorescence was measured at the end of eachextension step. After amplification, melting curves were acquired and used todetermine the specificity of PCR products, which were further confirmed usingconventional gel electrophoresis. The results were normalized against �-tubulin.

Statistical analysesQuantitative results are presented as the mean ± s.e.m. Differences between meanvalues of percentage of beating were calculated by using the Student’s t-test. Real-time PCR data were compared using analysis of variance (ANOVA) followed by aNewman-Keuls post-hoc test. Differences were considered significant when*P<0.05 and **P<0.001.

This work was supported by the research grant from the SwissNational Science Foundation (PMPDA-102408 to S.C.; 3100A0-109879 to C.C. and NRP 4046-058712 to M.J.) and from theSchmidheiny Foundation (Geneva). We thank Giulio Gabbiani for thecritical reading of the manuscript and Stephan Ryser for helpfuldiscussions and comments. We also gratefully acknowledge PaulaBorel, Giuseppe Celetta, Anita Hiltbrunner, Jean-Philippe Boquete

and Marie-Claude Belkouch for invaluable technical help, SergeiStartchik for help with the development of the Fourier transformationmacro, Serge Nef for embryo dissection, Wang Bei and Jian Li forteaching the hanging-drop method, and Constance Barazzone forproviding the mouse lung fibroblasts.

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